Homing peptides to receptors of heart vasculature

ABSTRACT

The present invention relates to peptides which selectively or preferentially home to areas of a heart. The invention further relates to conjugates of the homing peptides and uses thereof.

This application is a U.S. National Phase Application of InternationalApplication No. PCT/US2003/029379 filed on Sep. 18, 2003. Thespecification of International Application No. PCT/US2003/029379 ishereby incorporated by reference.

This application asserts priority to U.S. Provisional Application Ser.No. 60/412,330 filed on Sep. 19, 2002. The specification of U.S.Provisional Application Ser. No. 60/412,330 is hereby incorporated byreference.

The invention described in this application was made with finds from theNational Institutes of Health, Grant Number R01 AG20918-01. The UnitedStates government has certain rights in the application.

BACKGROUND OF THE INVENTION

The vasculature of a heart is comprised of blood vessels, such asarteries and veins, and the microvasculature. The microvasculatureincludes capillaries, arterioles, and venules, and are comprised ofmicrovasculature endothelial cells. Numerous receptors are present onthe cells that constitute the vasculature of a heart. The receptorsknown to exist on the cells include tumor necrosis factor (TNF)receptors and brain derived neurotrophic factor (BDNF) receptors.

Disease of the heart vasculature, i.e., cardiovascular disease, is theleading cause of morbidity and mortality in older individuals. As thepopulations ages, the need for optimal geriatric therapies is anincreasingly important public health issue. Currently, myocardialinfarction in older individuals has a significantly worse prognosis withhigher mortality and complication rates than younger individuals.Furthermore, depending, in part, on the extent of vascular damage, anindividual can have areas of the heart which are “old” (i.e., relativelyunhealthy) and other areas which are “young” (i.e., relatively healthy).Therefore, senescent changes in the cardiovascular system plays animportant role in predisposing older areas of the heart or older hearts,to increased vascular pathology.

Therefore, there is a need to determine the condition of a heart and toprovide treatments targeted to specific areas of a heart.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to an isolated peptide comprisingor consisting of the amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1).The peptide selectively homes to TNF receptor(s) in the vasculature of aheart.

In another embodiment, the invention relates to an isolated peptidecomprising or consisting of the amino acid sequence ARRGQAV (SEQ. ID.NO: 4). The peptide preferentially homes to BDNF receptor(s) in thevasculature of a heart.

In another embodiment, the invention relates to an isolated peptidecomprising or consisting of the amino acid sequence G(R/W)RFIRV (SEQ.ID. NO: 2). The peptide preferentially homes to BDNF receptor(s) in thevasculature of a heart.

In another embodiment, the invention relates to a conjugate comprising apeptide which comprises or consists of the amino acid sequenceQA(Q/E)GQLV (SEQ. ID. NO: 1) or functionally equivalent modificationsthereof, and a functional moiety. The peptide selectively homes to TNFreceptor(s) in the vasculature of a heart.

In another embodiment, the invention relates to a conjugate comprising apeptide which comprises or consists of the amino acid sequence ARRGQAV(SEQ. ID. NO: 4) or functionally equivalent modifications thereof, and afunctional moiety. The peptide preferentially homes to BDNF receptor(s)in the vasculature of a heart.

In another embodiment, the invention relates to a conjugate comprising apeptide which comprises or consists of the amino acid sequenceG(R/W)RFIRV (SEQ. ID. NO: 2) or functionally equivalent modificationsthereof, and a functional moiety. The peptide preferentially homes toBDNF receptor(s) in the vasculature of a heart.

In another embodiment, the invention relates to a method for determininga young heart or young areas of a heart vasculature in a mammal. Themethod comprises administering to the mammal a peptide comprising orconsisting of the amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1) orfunctionally equivalent modifications thereof, conjugated to adetectable marker, wherein the peptide selectively homes to TNFreceptor(s) in the vasculature of the heart and the marker is detected.A disproportionately high binding of the peptide comprising orconsisting of QA(Q/E)GQLV (SEQ. ID. NO: 1) indicates a young heart oryoung areas of a heart vasculature.

In another embodiment, the invention relates to a method for determiningan old heart or old areas of a heart vasculature in a mammal. The methodcomprises administering to the mammal a peptide comprising or consistingof the amino acid sequence ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ.ID. NO: 2) or functionally equivalent modifications thereof, conjugatedto a detectable marker, wherein the peptide selectively homes to BDNFreceptor(s) in the vasculature of the heart and the marker is detected.A disproportionately high binding of the peptide comprising orconsisting of ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID. NO: 2)indicates an old heart or old areas of a heart vasculature.

In another embodiment, the invention relates to a method for determiningthe condition of the vasculature of a heart in a mammal. The methodcomprises administering to the mammal a first peptide comprising orconsisting of the amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1) orfunctionally equivalent modifications thereof, conjugated to a firstdetectable marker. The first peptide selectively homes to TNFreceptor(s) in the vasculature of the heart. The method furthercomprises administering to the mammal a second peptide comprising orconsisting of the amino acid sequence ARRGQAV (SEQ. ID. NO: 4) orG(R/W)RFIRV (SEQ. ID. NO: 2) or functionally equivalent modificationsthereof, conjugated to a second detectable marker. The second peptidehomes to BDNF receptor(s) in the vasculature of the heart. The first andsecond marker are detected. A disproportionately high ratio of bindingof the first peptide to the second peptide indicates a young heart oryoung areas of the heart vasculature. Alternatively, adisproportionately low ratio of binding of the first peptide to thesecond peptide indicates an old heart or old areas of the heartvasculature.

In another embodiment, the invention relates to a method for deliveringa functional moiety selectively to a young heart or a young area of theheart in a mammal. The method comprises administering to the mammal aconjugate comprising a peptide which comprises or consists of the aminoacid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1) or functionally equivalentmodifications thereof, and a functional moiety.

In another embodiment, the invention relates to a method for deliveringa functional moiety preferentially to an old heart or an old area of theheart in a mammal. The method comprises administering to the mammal aconjugate comprising a peptide which comprises the amino acid sequenceARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID. NO: 2) or functionallyequivalent modifications thereof, and a functional moiety.

In another embodiment, the invention relates to a method for discoveringmimics of peptides comprising or consisting of amino acid sequenceQA(Q/E)GQLV (SEQ. ID. NO: 1) or functionally equivalent modificationsthereof. The method comprises determining a three-dimensional structureof peptides comprising or consisting of the sequence; identifyingcompounds comprising the structure; and determining the capacity of thecompounds for selective homing to TNF receptor(s) in the vasculature ofa heart. The compounds which home selectively to TNF receptor(s) in thevasculature of the heart are mimics.

In another embodiment, the invention relates to a method for discoveringmimics of peptides comprising or consisting of amino acid sequenceARRGQAV (SEQ. ID. NO; 4) or G(R/W)RFIRV (SEQ. ID. NO: 2) or functionallyequivalent modifications thereof. The method comprises determining athree-dimensional structure of peptides comprising or consisting of thesequence; identifying compounds comprising the structure; anddetermining the capacity of the compound for homing to BDNF receptor(s)in a vasculature of a heart. The compounds which home preferentially toBDNF receptor(s) in the vasculature of the heart are mimics.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid sequence of human TNFα Genbank Accession No. 1TNFA(SEQ. ID. NO: 6).

FIG. 2. Amino acid sequence of human BDNF Genbank Accession No. 1BNDA(SEQ. ID. NO: 7).

FIG. 3. Identification of aging-associated changes in cardiacmicrovascular epitope binding. (A) ψY12 epitope sequence homology toTNFα. (B) Homologous region is highlighted yellow in thethree-dimensional structure of TNFα. (C) Representative phageimmunostaining patterns in hearts with isolated helper phage and ψY12clones with minimal labeling of the 18-month old hearts (arrows). (D)ψY12 phage binding the cardiac microvasculature of 3-month old hearts(arrow). (E) ψY12 phage titers of 3- and 18-month old hearts.

FIG. 4. Aging-associated alterations in cardiac microvascular TNFαreceptor pathways. (A) Representative immunostaining for TNF receptor 1and 2 in the subepicardium and subendocardium of 3- and 18-month oldmurine hearts. (B) TNF receptor 1 and 2 densities in the subendocardiumof 3- and 18-month old murine hearts (n=3, each). (C) RT-PCR for PDGF-Binduction after TNFα treatment of 4- and 24-month old rat CMECs.

FIG. 5. Aging-associated alterations in TNFα cardioprotection. (A)Representative immunostaining for PDGF-B in rat hearts injected withTNFα or PBS vehicle 24 h prior to sacrifice. (B) Quantification ofPDGF-B density in the injected hearts (n=3, each group). (C)Representative cardiac histology after coronary occlusion: Mason'strichrome staining for myocardial necrosis (14 day post-coronaryocclusion) in 4- and 24-month old rat hears treated with PBS (4- and24-month old, n=4 and 10, respectively) and TNFα (4-month old, n=4); and(D) tunnel staining for apoptosis (DAB) with hematoxylin counter stain(2 day post-coronary occlusion) in 24-month old hearts treated with TNFα(n=10; 80% mortality vs. PBS 0%; p<0.05). (E) Quantification ofmyocardial necrosis in rat hearts 14 day post-coronary occlusion.

FIG. 6. Identification of aging-associated changes in BDNF homologousphage cardiac microvascular epitope binding. (A) ψO40 epitope sequencehomology to BDNF. (B) Homologous region is highlighted yellow in the 3-Dstructure of BDNF. (C) Representative phage immunostaining patterns inhearts with isolated ψO40 clones with phage binding the cardiacmicrovasculature of 18-month-old hearts with minimal binding to3-month-old hearts (n=3, each). (D) ψO40 phage titers of 3- and18-month-old hearts (n=3, each). *P<0.05 3-vs. 18-month-old hearts.

FIG. 7A-D. Aging associated alterations in cardiac microvascular BDNFreceptor pathways, immunostaining for the full-length receptor as wellas the truncated trkB receptor in sections of 3- and 18-month old ratheats.

FIG. 8. BDNF induction of activated macrophage recruitment in the heartsof old but not young rats. (A) Representative histology and immunostainsfor ED2 and ED1 in 4- and 24-month-old rat hearts injected with BDNF orPBS 24 hr prior to tissue harvest. (B) Quantification of ED1 and ED2cellular density in the injected hearts (n=3, each group). *P<0.05 BDNFvs. PBS.

FIG. 9. Aging-associated alterations in BDNF cardiac actions. (A)Representative cardiac histology after coronary occlusion: Masson'strichrome staining for myocardial necrosis (14 d post-coronaryocclusion) in 4- and 24-month-old rat hearts treated with PBS and BDNF.(B) Quantification of myocardial necrosis in rat hearts 14 dpost-coronary occlusion.

FIG. 10. Aging-associated BDNF cardiac aneurysm formation post coronaryocclusion. (A) Representative cardiac histology (Masson's trichrome)cardiac aneurysm formed in ⅗ 18-month-old rats receiving intramyocardialinjection of BDNF 1 d prior to coronary occlusion. (B) Inflammatoryinfiltrate in BDNF injected heart.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the surprising discovery that a peptide havingthe amino acid sequence QAQGQLV (SEQ. ID. NO: 3) binds to receptors inthe heart vasculature in a mammal, but does not bind significantly toreceptors on other organs. Similarly, a peptide having the amino acidsequence ARRGQAV (SEQ. ID. NO: 4) or GRRFIRV (SEQ. ID. NO: 5) binds toreceptors in the vasculature of the heart and brain, but do not bindsignificantly to receptors on other organs.

Also surprisingly, a peptide having the amino acid sequence QAQGQLV(SEQ. ID. NO: 3) binds with more binding capacity to TNF receptor(s) inthe vasculature of hearts which are “young” (i.e., healthy) than to TNFreceptor(s) in the vasculature of older hearts (i.e., unhealthy).Another discovery is that young hearts have more TNF receptor 1 presentthan do older hearts. The inventors also surprisingly discovered thattreatment of myocardial infarction with TNFα markedly reduced the extentof myocardial infarction in young hearts, but induced apoptosis ofcardiac cells in older hearts.

Furthermore, the inventors also discovered that a peptide having theamino acid sequence ARRGQAV (SEQ. ID. NO: 4) or GRRFIRV (SEQ. ID. NO: 5)bind to BDNF receptor(s) in the vasculature of older hearts, usually totrkB receptors, and more commonly to truncated trkB receptors. Anotherdiscovery is that older hearts have more truncated trkB receptors thando younger hearts. The inventors also surprisingly discovered thattreatment of myocardial infarction with BDNF had no effect on myocardialinfarctions in young hearts. In older hearts, BDNF increased the extentof myocardial infarction, induced inflammatory infiltration, andaneurysm formation.

As a result of the surprising discoveries described above, the presentinvention provides isolated peptides which selectively or preferentiallyhome to TNF receptor(s) or BDNF receptor(s) in the vasculature of aheart. Preferably, the homing peptides of the present invention bind toreceptors of the microvasculature of a heart, especially onmicrovasculature endothelial cells.

In one embodiment, a homing peptide comprises the amino acid sequenceQA(Q/E)GQLV (SEQ. ID. NO: 1) or a functionally equivalent modificationthereof. The amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 3) ishomologous to a sequence found at amino acid positions 16-22 of humanTNFα (FIG. 1). If the homing peptide comprises the amino acid sequenceQAQGQLV (SEQ. ID. NO: 3), the amino acid residues at positions 1-2 and4-6 are identical to the amino acid residues found at positions 16-17and 19-21 of human TNFα. If the homing peptide comprises the amino acidsequence QAEGQLV (SEQ. ID. NO: 8), the amino acid residues at positions1-6 are identical to the amino acid residues found at positions 16-20 ofhuman TNFα.

Homing peptides comprising the amino acid sequence QA(Q/E)GQLV (SEQ. ID.NO: 1) selectively home to TNF receptor(s) in the vasculature of aheart.

The TNF receptor(s) can be any receptor which binds TNFα. Examples ofTNF receptors include TNF receptor (TNFR) 1, TNFR2 (i.e., p75), andreceptors which are homologous to TNRF 1 or TNFR2, such as receptors inthe TNFR superfamily. Preferably, the TNF receptor is TNFR1.

In another embodiment, a homing peptide comprises the amino acidsequence ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID. NO: 2), or afunctionally equivalent modification thereof.

The amino acid sequence ARRGQAV (SEQ. ID. NO: 4) is homologous to asequence found at amino acid positions 5-11 of human BDNF (FIG. 2). Theamino acid residues at positions 1-5 of amino acid sequence ARRGQAV(SEQ. ID. NO: 4) are identical to the amino acids at positions 5-9 ofhuman BDNF (FIG. 2).

The amino acid sequence G(R/W)RFIRV (SEQ. ID. NO: 2) is homologous to asequence found at amino acid positions 99-105 of human BDNF. If thehoming peptide comprises the amino acid sequence GRRFIRV (SEQ. ID. NO:5), the amino acid residues at positions 1 and 3-6 are identical to theamino acid residues found at positions 99 and 101-104 of human BDNF(FIG. 2). If the homing peptide comprises GWRFIRV (SEQ. ID. NO: 9), theamino acid residues at positions 1-6 are identical to the amino acidresidues found at positions 99-104 of human BDNF.

Homing peptides comprising the amino acid sequence ARRGQAV (SEQ. ID. NO:4) or G(R/W)RFIRV (SEQ. ID. NO: 2) preferentially home to BDNFreceptor(s) in the vasculature of a heart.

The BDNF receptor(s) can be any receptor which binds BDNF. Examples ofBDNF receptors include, trkB, p75, and the nerve growth factor family ofreceptors, such as trkA and trkc. Usually, the BDNF receptor is trkB,and more commonly truncated trkB. “Truncated trkB” as used herein meansthat the trkB receptor lacks the intracellular tyrosine kinase domain.For example, the trkB receptor can lack the entire intracellulartyrosine kinase domain or can lack a portion of the domain. Typically,the truncated trkB receptor lacks a portion of the domain sufficient tocause loss of kinase activity.

The term “peptide” as used herein means an amino acid sequence having atleast five amino acid residues. No distinction will be made between apeptide, polypeptide, or protein. The peptide of the invention can belinear, cyclic, or branched.

The peptides of the invention include functionally equivalentmodifications of SEQ. ID. NOs: 1, 2, 3, 4, 5, 8 and 9. A functionallyequivalent modification refers to a molecule having a similar,non-identical sequence and the selective or preferential homing propertydescribed herein. A functionally equivalent modification can be, forexample, two, preferably one deletions and/or substitutions relative tothe reference peptide sequence. Any deletions or substitutions in theamino acid sequence of the reference peptide are permitted provided thatthe peptide of the invention continues to satisfy the functionalcriteria described above and below.

Substitutions in the amino acid sequence of the reference peptide arepreferably with equivalent amino acids. Groups of amino acids known tobe of equivalent character are listed below:

-   -   (a) Ala(A), Ser(S), Thr(T), Pro(P), Gly(G);    -   (b) Asn(N), Asp(D), Glu(E), Gln(O);    -   (c) His(H), Arg(R), Lys(K);    -   (d) Met(M), Leu(L), Ile(I), Val(V); and    -   (e) Phe(F), Tyr(Y), Trp(W).

The deletions can include any one or two amino acid residue deletions.Preferably, the deletions are one or two amino acid residues at eitherthe N- or C-terminal of the reference peptide. Most preferably, thedeletions are the amino acid residues that are not homologous to TNFα orBDNF.

Therefore, in this specification, when reference is made to amino acidsequences QA(Q/E)GQLV (SEQ. ID. NO: 1), ARRGQAV (SEQ. ID. NO: 4), orG(R/W)RFIRV (SEQ. ID. NO: 2), the functionally equivalent modificationsare also included.

Preferred amino acid residues for the functionally equivalentmodifications of the peptide comprising the amino acid sequenceQA(Q/E)GQLV (SEQ. ID. NO: 1) are a Q at amino acid position 3 or a V atamino acid position 7. In an especially preferred embodiment, Q ispresent at amino acid position 3 and V is present at amino acid position7.

Similarly, preferred amino acid residues for the functionally equivalentmodifications of the peptide comprising the amino acid sequence ARRGQAV(SEQ. ID. NO: 4) are an A at amino acid position 6 or a V at amino acidposition 7. In an especially preferred embodiment, A is present at aminoacid position 6 and V is present at amino acid position 7.

Likewise, preferred amino acid residues for the functionally equivalentmodifications of the peptide comprising the amino acid sequenceG(R/W)RFIRV (SEQ. ID. NO: 2) are a W at amino acid position 2 or a V atamino acid position 7. In an especially preferred embodiment, W is atamino acid position 2 and V is at amino acid position 7.

The homing peptides described above can further comprise additionalamino acid residues. The additional amino acids residues can be anynumber such that the peptide selectively or preferentially homes to thereceptor(s) described above in the vasculature of a heart. For instance,the homing peptide can be a relatively short peptide. The approximateminimum number of amino acid residues in the peptide can, for example,be 5, 7, 10, 15, 20, 30, or 40 amino acids in length. There is noparticular maximum number of amino acid residues for the homing peptidesof the peptide invention. For example, a suitable maximum number ofamino acid residues in the peptide can be approximately 200, morepreferably approximately 175, and even more preferably approximately 150amino acid residues.

The additional amino acid residues can be any amino acid residue suchthat the peptide selectively or preferentially homes to the receptor(s)described above in the vasculature of a heart. For example, if thehoming peptide comprises the amino acid sequence QA(Q/E)GQLV (SEQ. ID.NO: 1), the additional amino acid residues can be any one or more aminoacid residues that occur naturally at the N-terminal and/or C-terminalside of amino acid residues at positions 16-22 of human TNFα (FIG. 1).

For instance, naturally occurring amino acid resides which can be foundN-terminal to amino acid residues at positions 16-22 of human TNFαinclude the amino acid residues approximately at positions 10-15, 5-15,and 2-15 of human TNFα. Similarly, naturally occurring amino acidresides which can be found C-terminal to amino acid residues atpositions 16-22 of human TNFα include the amino acid residuesapproximately at positions 23-60, 23-120, and 23-150 of human TNFα.

Amino acid residues approximately at positions 10-15, 5-15, and 2-15 ofhuman TNFα are preferably attached to the N-terminus of QA(Q/E)GQLV(SEQ. ID. NO: 1). The amino acid residues approximately at positions23-60, 23-120, and 23-150 are preferably attached to the C-terminus ofQA(Q/E)GQLV (SEQ. ID. NO: 1). It is not necessary to have additionalamino acid residues at either or at both the N- and C-terminus ofQA(Q/E)GQLV (SEQ. ID. NO: 1). For example, the additional amino acidresidues can be attached just at the N-terminus of QA(Q/E)GQLV (SEQ. ID.NO: 1) or just at the C-terminus, or both. In addition, the homingpeptide can have no additional amino acid residues attached. Thus, thehoming peptide can consist of the amino acid sequence QAQGQLV (SEQ. ID.NO: 3) or QAEGQLV (SEQ. ID. NO: 8).

If the homing peptide comprises the amino acid sequence ARRGQAV (SEQ.ID. NO: 4), the additional amino acid residues can be any one or moreamino acid residues that occur naturally at the N-terminal and/orC-terminal side of amino acid residues at positions 5-11 of human BDNF(FIG. 2). For example, naturally occurring amino acid resides which canbe found N-terminal to amino acid residues at positions 5-11 of BDNFinclude the amino acid residues approximately at positions 1-4 and 3-4of BDNF. Similarly, naturally occurring amino acid resides which can befound C-terminal to amino acid residues at positions 5-11 of human BDNFinclude the amino acid residues approximately at positions 12-30, 12-50,12-70, 12-90, and 12-110 of BDNF.

Amino acid residues approximately at positions 1-4 or 34 of human BDNFare preferably attached to the N-terminus of ARRGQAV (SEQ. ID. NO: 4).The amino acid residues approximately at positions 12-30, 12-50, 12-70,12-90, or 12-110 are preferably attached to the C-terminus of ARRGQAV(SEQ. ID. NO: 4). It is not necessary to have additional amino acidresidues at either or at both the N- and C-terminus of ARRGQAV (SEQ. ID.NO: 4). For example, the additional amino acid residues can be attachedjust at the N-terminus of ARRGQAV (SEQ. ID. NO: 4) or just at theC-terminus, or both. In addition, the homing peptide can have noadditional amino acid residues attached. Thus, the homing peptide canconsist of the amino acid sequence ARRGQAV (SEQ. ID. NO: 4).

If the homing peptide comprises the amino acid sequence G(R/W)RFIRV(SEQ. ID. NO: 2), the additional amino acid residues can be any one ormore amino acid residues that occur naturally at the N-terminal and/orC-terminal side of amino acid residues at positions 99-105 of human BDNF(FIG. 2). For example, naturally occurring amino acid resides which canbe found N-terminal to amino acid residues at positions 99-105 of BDNFinclude the amino acid residues approximately at positions 80-98, 60-98,40-98, 20-98, and 4-98 of BDNF. Similarly, naturally occurring aminoacid resides which can be found C-terminal to amino acid residues atpositions 99-105 of human BDNF include the amino acid residuesapproximately at positions 106-109, 106-112, and 106-115 of BDNF.

Amino acid residues approximately at positions 80-98, 60-98, 40-98,20-98, or 4-98 of human BDNF are preferably attached to the N-terminusof G(R/W)RFIRV (SEQ. ID. NO: 2). The amino acid residues approximatelyat positions 106-109, 106-112, or 106-115 are preferably attached to theC-terminus of G(R/W)RFIRV (SEQ. ID. NO: 2). It is not necessary to haveadditional amino acid residues at either or at both the N- andC-terminus of G(R/W)RFIRV (SEQ. ID. NO: 2). For example, the additionalamino acid residues can be attached just at the N-terminus ofG(R/W)RFIRV (SEQ. ID. NO: 2) or just at the C-terminus, or both. Inaddition, the homing peptide can have no additional amino acid residuesattached. Thus, the homing peptide can consist of the amino acidsequence GRRFIRV (SEQ. ID. NO: 5) or GWRFIRV (SEQ. ID. NO: 9).

The peptides of the invention, including the functionally equivalentmodified peptides, are isolated. An isolated peptide is substantiallyfree from other biological components, as well as from materials thatmay be used in preparation, isolation, characterization or purificationof peptides. Examples of other biological components include cellularcomponents, culture media or components (including conditioned media andcomponents thereof, including proteins and nucleic acid molecules),affinity binding agents, such as immunoconjugates or antibodies andother serum components. Examples of materials that may be used inpreparation, isolation, or purification of proteins include separationmedia or membranes, such as nitrocellulose, chromatographic matrices,and electrophoretic gel media, including for instance, polyacrylamideand detergents, such as sodium dodecyl sulfate (SDS).

Preferably, the isolated peptide is at least about 25% to about 90%pure, i.e., free from other peptides and nucleic acid molecules. Morepreferably, the isolated material is at least about 50% to about 90%pure. Optimally, the isolated peptide is at least about 75% to about 90%pure.

Most preferably, the peptides of the invention, including thefunctionally equivalent modified peptides, are purified. As used herein,the term “purified” means essentially pure as demonstrated by singleband purity on electrophoresis in SDS-polyacrylamide gels (SDS PAGE).Preferably, the purified material is at least about 90% to about 99.9%pure. More preferably, the purified material is at least about 95% toabout 99.9% pure. Optimally, the purified material is at least about 99%to about 99.9% pure.

Peptides comprising QA(Q/E)GQLV (SEQ. ID. NO: 1) selectively home to TNFreceptor(s) in the vasculature of a heart. A homing peptide is selectivefor TNF receptor(s) in the heart vasculature if the peptide bindsdisproportionately to TNF receptor(s) in the heart vasculature, i.e.,binds at least two-fold greater to TNF receptor(s) in the heartvasculature than to TNF receptor(s) in non-heart vasculature, such asfor example, TNF receptor(s) in the vasculature of the brain or kidney.Preferably, the homing peptide binds at least five-fold greater, andmore preferably at least ten times greater to TNF receptor(s) in theheart vasculature than to TNF receptor(s) in non-heart vasculature.

Selective homing can be demonstrated by determining if binding of ahoming peptide to TNF receptor(s) in the heart vasculature is specificin accordance with the above definition. For example, the amount of aparticular homing peptide bound to TNF receptor(s) in the heartvasculature can be compared to the amount of homing peptide which bindsto TNF receptor(s) in non-heart vasculatures. Selective homing can alsobe determined by showing that peptides that home to the TNF receptor(s)in the heart vasculature are enriched in one or more subsequent roundsof in vivo panning.

Peptides comprising ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID.NO: 2) preferentially home to BDNF receptor(s) in the vasculature of aheart. A homing peptide is preferential for BDNF receptor(s) in theheart vasculature if the peptide binds disproportionately to BDNFreceptor(s) in the vasculature of the heart or brain, i.e., binds atleast two-fold greater to BDNF receptor(s) in the heart or brainvasculature than to BDNF receptor(s) in non-heart or non-brainvasculatures, such as for example, BDNF receptor(s) in the vasculatureof the kidney. Preferably, the homing peptide binds at least five-foldgreater, and more preferably at least ten times greater to BDNFreceptor(s) in the heart or brain vasculature than to BDNF receptor(s)in non-heart or non-brain vasculature. Binding of the peptide to thevasculature of an organ can be determined by, for example, a bindingassay.

The peptides of the present invention can be conjugated to a functionalmoiety. Due to the selective or preferential homing of the peptides ofthe present invention to TNF receptor(s) or BDNF receptor(s) in thevasculature of a heart, the functional moiety can be delivered toappropriate areas of the vasculature of a heart. The conjugates can beused to determine the quality (health status) of a heart and/or todeliver therapeutic agents to the appropriate areas of a heart.

In one embodiment, the functional moiety is a detectable marker. Themarker can be any marker known to those in the art. For example, themarker can be any marker which emits a detectable signal. Examples ofmarkers include radioisotopes, radionuclides, and fluorophores. Anexample of a fluorophore include radiofluoronated compounds. Examples ofradioisotopes include thallium, iodine¹²⁵, and radioopaque contrastagents.

The markers can be detected by any known method in the art. The methodfor detecting the marker depends on the marker used. For example, if themarkers are radiofluoronated compounds, the markers can be detected byPositron Emission Tomography (PET) scan. If the markers areradioisotopes, the marker can be detected with, for example, a gammacamera. The detectable marker generally allows for detection,visualization, or imaging of the vasculature of a heart.

The homing peptides conjugated to markers can be used to determine thecondition of a heart. For example, peptides comprising amino acidsequence QA(Q/E)GQLV (SEQ. ID. NO: 1) bind disproportionately to TNFreceptor(s) in the vasculature of a young heart or young portions of aheart vasculature. Disproportionate detection of a marker conjugated toa homing peptide comprising the amino acid sequence QA(Q/E)GQLV (SEQ.ID. NO: 1) indicates a young heart or areas of a heart which are young.

Similarly, peptides comprising amino acid sequence ARRGQAV (SEQ. ID. NO:4) or G(R/W)RFIRV (SEQ. ID. NO: 2) bind disproportionately to BDNFreceptor(s) in the vasculature of an old heart or old portions of aheart vasculature. Disproportionate detection of a marker conjugated toa homing peptide comprising the amino acid sequence ARRGQAV (SEQ. ID.NO: 4) and/or G(R/W)RFIRV (SEQ. ID. NO: 2) indicates an old heart orareas of the heart which are old.

The terms “young” and “old” as used in this specification, do not refernecessarily to the age of the individual. Instead “young” and “old”refer to the condition of the vasculature of a heart. A youngindividual, for example, can have a heart which is “old” or haveportions of the heart which are “old.” In contrast, an old individualcan have a heart which is “young” or have portions of the heart whichare “young.” Young hearts or young portions of a heart vasculature aretypically healthy. Old heart or old portions of a heart vasculature aretypically unhealthy or damaged.

The disproportionate capacity of a heart or portions of a heartvasculature to bind a peptide comprising the amino acid sequenceQA(Q/E)GQLV (SEQ. ID. NO: 1) indicates a young heart or young portionsof a heart vasculature. The disproportionate capacity of a heart orportions of a heart vasculature to bind a peptide comprising the aminoacid sequence ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID. NO: 2)indicates an old heart or old portions of a heart vasculature.

Administration to a mammal of a peptide comprising the amino acidsequence QA(Q/E)GQLV (SEQ. ID. NO:1) conjugated to a detectable markercan be used to determine whether a heart is young or has young areas.Detection of the marker indicates the capacity of the heart or areas ofthe heart vasculature to bind TNFα. A disproportionately high binding ofthe peptide comprising amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO:1)is indicative of a young heart or young areas of a heart vasculature. Incontrast, a disproportionately low binding indicates an old heart or oldareas of a heart vasculature.

To determine an old heart or old areas of a heart, a peptide comprisingthe amino acid sequence ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ.ID. NO: 2) conjugated to a detectable marker is administered to amammal. The capacity of the heart or portions of the heart vasculatureto bind BDNF can be determined by detecting the marker. Adisproportionately high binding of the peptide comprising the amino acidsequence ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID. NO: 2) isindicative of an old heart or old areas of a heart vasculature. Incontrast, a disproportionately low binding indicates a young heart oryoung areas of a heart vasculature.

Optimally, to determine the condition of a heart, a peptide comprisingthe amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1) conjugated to adetectable marker and a peptide comprising either the amino acidsequence ARRGQAV (SEQ. ID. NO: 4) or G(R/W)RFIRV (SEQ. ID. NO: 2)conjugated to another detectable marker are administered to a mammal. Adisproportionately high ratio of binding of the peptide comprising theamino acid sequence QA(Q/E)GQLV (SEQ. ID. NO:1) to binding of thepeptide comprising either the amino acid sequence ARRGQAV (SEQ. ID. NO:4) or G(R/W)RFIRV (SEQ. ID. NO: 2) indicates a young heart or youngareas of a heart vasculature. A disproportionately high ratio is greaterthan two, more preferably greater than three, and even more preferablygreater than four. Alternatively, a disproportionately low ratio (i.e.,ratio less than 2) indicates an old heart or old areas of a heartvasculature.

The order of administration of the peptides is not important indetermining the condition of a heart. For example, the selective homingpeptide comprising QA(Q/E)GQLV (SEQ. ID. NO: 1) conjugated to adetectable marker can be administered before, after, or simultaneouslywith the preferential homing peptide comprising the amino acid sequenceARRGQAV (SEQ. ID. NO: 4) or Q(R/W)RFIRV (SEQ. ID. NO: 2) conjugated toanother detectable marker.

Furthermore, the homing peptides conjugated to a detectable marker canalso be used to determine the prognosis of cardiac therapy. For example,an image obtained of a heart vasculature before therapy begins, which isindicative of its condition, can be compared to an image obtained aftertherapy begins. A decrease in the old areas of a heart vasculature or anincrease in the young areas indicates an effective therapy.

In another embodiment, the functional moiety is a therapeutic agent. Thetherapeutic agent can be a small molecule or a biological molecule. Abiological molecule is any molecule which contains a nucleic acid oramino acid sequence and has a molecular weight greater than 450.

Biological molecules which can be used as therapeutic agents includeviral gene therapy vectors; viruses; nucleic acid molecules andoligonucleotides including antisense and dominant negative molecules;polypeptides, peptides, and proteins. Examples of proteins includemicrovasculature growth factors such as platelet derived growth factor,vascular endothelial growth factor, angiopoietin, and estrogen.

Small molecules include organic compounds, organometallic compounds,salts of organic and organometallic compounds, saccharides, amino acids,and nucleotides. Small molecules can further include molecules thatwould otherwise be considered biological molecules, except theirmolecular weight is not greater than 450. Thus, small molecules may belipids, oligosaccharides, oligopeptides, and oligonucleotides, and theirderivatives, having a molecular weight of 450 or less.

It is emphasized that small molecules can have any molecular weight.They are merely called small molecules because they typically havemolecular weights less than 450. Small molecules include compounds thatare found in nature as well as synthetic compounds.

In another embodiment, the invention provides methods for delivering afunctional moiety to a heart which contain young areas or hearts.Functional moieties, such as detectable markers, can be delivered asdescribed above. The function moiety can also be a therapeutic agent.The therapeutic agent can be used to treat young hearts or young areasof a heart in a cardiac pathology.

Therapeutic agents are targeted to young areas of a heart byadministering an appropriate therapeutic agent conjugated to a peptidecontaining the amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1).Therefore, the therapeutic agent is selectively delivered to theappropriate areas with reduced adverse effects on other organs or areasof the heart.

For example, young areas or hearts can be treated in order to induceproliferation of the cells in the microvasculature of the heart. Theproliferation of the young cells of the microvasculature in the heartcan compensate for areas damaged by a cardiac pathology.

Therapeutic agents which are useful to treat young areas of a heart oryoung hearts are known to those in the art and include the therapeuticagents described herein. Therapeutic agents which induce proliferationof heart microvascular cells include, for example, growth factors, suchas platelet derived growth factor, vascular endothelial growth factor,and angiopoietin.

In another embodiment, a functional moiety can be delivered to a heartcontaining old areas or hearts. Functional moieties, such as detectablemarkers, can be delivered as described herein. The function moiety canalso be a therapeutic agent. The therapeutic agent can be used to treatold hearts or old areas of a heart in a cardiac pathology.

Therapeutic agents are targeted to the old area of a heart byadministering an appropriate therapeutic agent conjugated to a peptidecontaining the amino acid sequence ARRGQAV (SEQ. ID. NO: 4) orG(R/W)RFIRV (SEQ. ID. NO: 2). Therefore, the therapeutic agent ispreferentially delivered to areas damaged by a cardiac pathology.

Therapeutic agents which are useful to treat old areas of a heart in aparticular cardiac disease are known to those in the art. Thetherapeutic agents include, for example, agents that inhibit apoptosisin old hearts or old areas of hearts. Such agents include, for example,estrogen.

Any cardiac pathology that results in damage to a heart can be treatedin accordance with the present invention. Cardiac pathologies include,for example, myocardial infarction, myocardial hypertrophy, congenitalheart disease, ischemic heart disease, and heart failure.

The conjugates are administered to a mammal in an effective amount. An“effective amount” is the amount of the conjugate that produces adesired effect. An effective amount will depend, for example, on thefunctional moiety conjugated to the homing peptide, the pathology beingtreated, and the age, size, and condition of the mammal. An effectiveamount of a particular conjugate for a particular cardiac pathology isknown to, or can routinely be determined by, those skilled in the art.

The conjugate can be administered to a mammal by various routes,including, for example, orally, systemically, or parenterally, such asintravenously. The conjugate can be administered by injection or byintubation.

A mammal can be any mammal. Examples of mammals include primates, suchas humans, laboratory animals, such as rats and mice, pet animals, suchas dogs and cats, and farm animals, such as cows and sheep.

A conjugate is generally delivered as a pharmaceutical composition. Thepharmaceutical composition can, for example, contain a pharmaceuticallyacceptable carrier. Such carriers are well known in the art and include,for example, aqueous solutions such as physiologically buffered salineor other solvents or vehicles such as glycols, glycerol, and oils suchas olive oil.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the complex. Such physiologically acceptable compoundsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. One skilled in the art knows that the choice of apharmaceutically acceptable carrier, including a physiologicallyacceptable compound, depends, for example, on the route ofadministration of the composition.

In another embodiment, mimics of peptides comprising amino acidsequences QA(Q/E)GQLV (SEQ. ID. NO: 1), ARRGQAV (SEQ. ID. NO: 4), orG(R/W)RFIRV (SEQ. ID. NO: 2) are discovered. A mimic is any compoundhaving the same three-dimensional structure and binding property aspeptides having amino acid sequence QA(Q/E)GQLV (SEQ. ID. NO: 1),ARRGQAV (SEQ. ID. NO: 4), or G(R/W)RFIRV (SEQ. ID. NO: 2).

The three-dimensional structure of a peptide can be determined by anyknown method in the art. Examples of methods for determining thethree-dimensional structure of a peptide include, x-ray crystallographyand computational methods, such as 3-D structure prediction programsbased on, for example, homology modeling. Examples of such programsinclude SWISS-MODEL, which is available athttp://www.expasy.ch/swissmod/SWISS-MODEL.html, and CPHmodels, which isavailable at http://www.cbs.dtu.dk/services/CPHmodels/.

Compounds comprising the three-dimensional structure of the homingpeptides of the present invention can be identified by any known methodin the art. Compounds can be identified by, for example, binding assays,competition assays, spectrophotometry, and x-ray crystallography. Thecompound can be a small molecule or a biological molecule. The selectiveand preferential homing capacity of the compounds can be determined asdescribed above. Those compounds which selectively or preferentiallyhome to TNF receptor(s) or BDNF receptor(s) in the vasculature of theheart are mimics.

The peptides of the invention and their mimics can act as an agonist oran antagonist depending on the ability or inability of the peptide orthe mimic to activate the receptor. An agonist generally binds to areceptor and stimulates the function of the receptor. A peptide or mimicthat activates TNF receptor(s) or BDNF receptor(s) is an agonist.Methods to determine whether a peptide or mimic activates TNFreceptor(s) or BDNF receptor(s) are known to those skilled in the art.

An antagonist generally binds to a receptor and inhibits activation ofthe receptor. A peptide or mimic that inhibits the activation of TNFreceptor(s) or BDNF receptor(s) is an antagonist. Method to determinewhether a peptide or mimic inhibits the activation of TNF receptor(s) orBDNF receptor(s) are known to those skilled in the art.

An agonist can be useful, for example, in treating myocardial infarctionof a young heart or young areas of a heart. Activation of TNFreceptor(s) by the agonist can reduce the extent of myocardial damage tothe heart.

In contrast, an antagonist can be used to inhibit the activation of TNFreceptor(s), for example, to inhibit apoptosis of old hearts or oldareas of a heart.

Similarly, an antagonist can be used to inhibit the activation of BDNFreceptor(s) in old hearts of old areas of a heart, thereby preventing,for example, infiltration of inflammatory cells and aneurysm formation.

EXAMPLES Example 1 ψY12 with Homology to TNFα Binds to Hearts in 3-MonthOld Mice

Cardiac-homing phage clone ψY12 (SEQ ID NO: 10) containing the aminoacid sequence QAQGQLV (SEQ ID NO: 1) (FIGS. 3A and 3B), which ishomologous to TNFα (SEQ ID NO: 11), identified an aging-associatedchange in TNFα receptor pathways in the microvasculature of the olderheart. In vivo injection of the ψY12 clone phage confirmed thediminished binding capacity of the TNFα-like phage in the older cardiacmicrovasculature (FIGS. 3C-3E), with only minimal binding in thesubepicardium of aging hearts. Therefore, TNF receptor is altered in themicrovasculature of the older heart.

Example 2 TNF Receptor Expression in Microvasculature of Young and OldMice

Immunostaining of 3- and 18-month old hearts revealed TNF receptor 2 inthe microvasculature throughout the young and older hearts (FIGS. 4A and4B). TNF receptor 1 was present in the microvasculature throughout theyoung hearts, however, in the older hearts, the receptor was restrictedto the subepicardial microvasculature, consistent with the patterning ofψY12 in Example 1.

Example 3 Functional Significance of the Change in TNF Receptor in AgingCardiac Microvasculature

The functional significance of the changes in TNF receptor in the agingcardiac microvasculature was examined in vitro. Cardiac microvascularendothelial cells (CMECS) of 4 and 24-month old F344 rats were isolatedand cultured. Briefly, the hearts were removed and minced in endothelialcell medium (DMEM containing 20% FBS, 1% endothelial cell growth factor,1% endothelial cell growth supplement, 1% BME, 10⁻⁴% heparin and 1%penicillin [10,000 I.U./ml]/streptomycin [10,000 μg/ml]), and digestedwith medium containing 0.2% collagenase and 0.005% Dnase, and 5% FBS for45 minutes with subsequent endothelial cells isolation by PECAM-mediatedmagnetic particle collection (Dynabeads). The resultant CMECs (Dil-LDLuptake>95%) were cultured for 2 passages and seeded into 12-well culturedish (10⁵ cells/well) and grown to confluence. Medium was changed toserum free-DMEM for 1 hr and then to medium containing 2% FBS+TNFα (30ng/ml) for 3 hr.

In vitro, TNFα induced platelet derived growth factor (PDGF)-Bexpression in cardiac microvascular endothelial cells (CMECs) from theyoung, but not in older hearts (FIG. 4C).

Example 4 In Vivo Response to TNFα

To examine the age-dependent effects of TNFα PDGF-B expression andprotection from myocardial necrosis, sets of 4- and 24-month old F344rats received intramyocardial injections of growth factor. Briefly, therats were anesthetized and underwent left intercostals thoracotomy.After identifying the left anterior descending artery (LAD), 100 ng ofTNFα in 50 μl PBS or PBS alone was injected through a 30 G needle usinga 250 μl Hamilton syringe. Two injections (25 μl/injection 2 mm apart)were made at mid ventricular anterior wall. The chest wall was thenclosed, the lungs inflated, the rat extubated, and the tracheotomyclosed.

In vivo, TNFα specifically induced increase of PDGF-B in the younghearts, while having minimal effect on the aging tissue (FIGS. 5A and5B). Moreover, TNFα markedly reduced the extent of myocardial infarctionin the young hearts (FIGS. 5C and 5D). In the older rats TNFα washarmful, inducing extensive apoptosis in the anterior left ventricularwall and resulting in an 80% mortality within 72 h of coronaryocclusion.

Example 5 ψO40 with Homology to BDNF Binds to Heart in Older Mice

Cardiac-homing phage clone ψO40 (SEQ ID NO: 12) containing the aminoacid sequence ARRGQAV (SEQ ID NO: 4), which is partially homologous toBDNF (SEQ ID NO: 13) (FIGS. 6A and 6B), identified an aging-associatedchange in BDNF receptor pathways in the microvasculature of the olderheart. In vivo injection of the ψO40 clone phage combining withimmunostaining and recovered ψO40 phage CFU assay confirmed the morebinding capacity of the BDNF-homologous phage in the older cardiacmicrovasculature while only minimal binding of the subepicardium of theyoung hearts, suggesting that BDNF receptor(s) may be altered in themicrovasculature of the older hearts (FIGS. 6C and 6D).

Indeed, immunostaining of the BDNF receptor, trkB, demonstrated that thedensity of the truncated trkB^(trk) receptor was significantly greaterin the hearts of older mice and rats compared with the hearts of youngermice and rats, while the distribution of the full-length receptor wassimilar in both age groups (FIG. 7A-D).

Example 6 Functional Significance of the Change in BDNF Receptor inAging Cardiac Microvasculature

To probe the age-dependent effects of BDNF on the aging heart, sets of4- and 24-month old F344 rats (n=3, each set) received intramyocardialinjections of the growth factor as described in Edelberg et al. (Circul2002, 105:608-613). Briefly, the rats were anesthetized and underwentleft intercostals thoracotomy. After the left anterior descending artery(LAD) was identified, 1 μg of rhBDNF (R&D Systems, 248BS005) in 50 μl ofPBS or PBS alone was injected through a 30 G needle using a 250 μlHamilton syringe. Two injections (25 μl/injection 2 mm apart) were madeat mid-left ventricular anterior wall. The chest wall was then closed,the lungs inflated, the rat extubated, and the tracheotomy closed.

Rats receiving pretreatment alone (BDNF or control) were sacrificed 24hr postinjection. The hearts were excised, fixed, sectioned,immunostained for ED1 and ED2, and visualized with DAB. Staining wasquantified in the anterior and posterior left ventricular wall at thelevel of the mid-papillary muscles from each heart, as described inEdelberg et al. (Circul. 2002, 105:608-613) [ten high power fields (40×per heart]. Two investigators performed quantification independently ina blinded fashion.

The functional significance of the alteration in BDNF receptor pathwayin the aging cardiac microvaculature was examined in vivo.Intramyocardail injection of BDNF 24 hr prior to sacrifice resulted in asimilar infiltration of macrophage in both young and older rat hearts,as evidenced by histology and immunostaining for ED2, FIG. 8. However,in the older heart BDNF-mediated induction of activated/inflammatorymacrophage was significantly greater compared with young hearts, asevidences by immunostaining for ED1.

Example 7 In Vivo Response to BDNF in Young and Old MyocardialInfarction Hearts

The potential effects of BDNF pretreatments were studied in a myocardialinfarction model. One day after intramyocarial injection of BDNF(4-month old, n=5; 24-month old, n=5) or control (4-month old, n=5;24-month old, n=5), the rats were anesthetized, the heart exposed, andthe LAD ligated just below (4-month old) or 2 mm below (24-month old, toproduce a similar size myocardial infarction to the younger rats) theleft atrial appendage with 8-0 nylon sutures. Pallor and regional wallmotion abnormality of the left ventricle confirmed occlusion. The chestwall was closed and, after recovery, the rats were returned to theanimal facility for 14 d. At the termination of the experiment, the ratswere sacrificed and the hearts explanted. The extent of myocardialinfarction measured at the level of the mid-papillary heart muscles wasscored by Masson's trichrome staining, and the images were analyzed in ablinded fashion employing ImageJ 1.22 software (NIH Image). Infarctionsize was expressed as a percentage of the total left ventriclemyocardial area. Differences were tested for statistical significance bythe Wilcoxon rank-sum test. P value<0.05 was considered significant.

In myocardial infarction studies, BDNF demonstrated a similarage-associated effect on cardiac physiology. In the young heart,intramyocardial injection of BDNF 24 hr before coronary occlusion didnot alter myocardial infarction size compared with PBS controls (FIG.9). In the older rats, BDNF was deleterious, with significantly moreextensive myocardial infarctions compared with hearts injected withcontrol. Moreover, BDNF injection resulted in the formation ofventricular wall aneurysms (3/5 rats) with persistent macrophageinfiltrates two weeks after coronary occlusion (FIG. 10).

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled, “sequence_listing2.txt”,created on Jan. 5, 2011. The sequence.txt file is 4.74 kb in size.

1. A method for delivering a functional moiety to a trkB receptor in theheart microvasculature in a mammal, the method comprising administeringa conjugate, said conjugate comprising a peptide comprising the aminoacid sequence of SEQ ID NO: 4 and said functional moiety.
 2. A methodaccording to claim 1, wherein said functional moiety is a therapeuticagent.
 3. A method according to claim 2, wherein said therapeutic agentis estrogen.
 4. A method according to claim 1, wherein said functionalmoiety is a detectable marker.